1. Field of the Invention
This invention is related to the quantitative analysis of active species in solution. It is particularly useful for analyses of proteins in solutions containing interfering species, for example, effluent solutions from the ion exchange chromatographic separation of proteins. This invention is a result of a contract with the United States Department of Energy.
Separation of proteins by ion exchange chromatography is receiving considerable attention in the field of clinical diagnostics. Separation and analysis of isoenzymes (enzymes which catalyze the same reaction) have become important in diagnostic procedures with the discovery that various tissues express different isoenzyme contents. By monitoring isoenzyme concentration in serum one can identify tissue damage by a non-invasive method. For example, certain isoenzymes of creatine kinase (CK) and lactate dehydrogenase (LD) have molecular forms found predominantly in heart tissue. The quantitative analysis of creatine kinase and lactate dehydrogenase in body fluids such as serum can play a major role in the verification of myocardial infarction.
Lactate dehydrogenase activity is expressed by five isoenzymes, LD-1 through LD-5. Elevations in LD-1 and LD-2 suggest cardiac damage while elevations in LD-4 and LD-5 are indicative of hepatic or muscular damage. Three isoenzymes of creatine kinase have been identified; CK-MM, CK-MB, and CK-BB. The primary source of CK-MM, CK-MB, and CK-BB is, respectively, skeletal muscle, myocardium and smooth muscle. CK-MB has been reported to have a zero incidence of false negative and a reported specificity of 99% for myocardial infarction. See Wagner, G. S., et al, "The Importance of the Identification of the Myocardial-Specific Isoenzyme of Creatine Phosphokinase (MB form) in the Diagnosis of Acute Myocardial Infarction," Circulation, Vol. 47, p. 263 (1973). The elevation in concentration of CK-MB in serum is short-lived, however, rising acutely within the four hours after an infarction, peaking within 24 hours, and then returning to normal within 48 hours. Fortunately, an increase in the serum levels of LD-1 and LD-2 occurs about 12-24 hours after the onset of infarction symptoms and persists for days thereafter. Although lactate dehydrogenase isoenzymes are not as specific as CK-MB, for heart damage, the two assays taken in conjunction are confirmatory and virtually 100% diagnostic of myocardial infarction. The present invention is particularly useful for providing these and other diagnostic isoenzyme analyses.
2. Description of the Prior Art
In describing the work of others herein, we do not admit that such work is actually prior art under 35 USC 102 or 35 USC 103 or that the work was prior in time to the making of the invention described and claimed herein. We reserve the right to establish a date of conception and reduction to practice prior to the effective date of any publication or work herein described.
A number of writers have described the separation of isoenzymes by high performance chromatography and electrophoresis, see for example, Mercer, D. W., "Simultaneous Separation of Serum Creatine Kinase and Lactate Dehydrogenase Isoenzymes by Ion-Exchange Column Chromatography, "Clinical Chemistry, Vol. 21, No. 8, pp. 1102-1106 (1975). Selective photometric determination of isoenzymes in protein solutions has been impractical. Isoenzymes, as do most other proteins, absorb at 280 nm precluding selective or comparative absorption analyses in the presence of other proteins.
Schroeder, R. R., et al in "Enzyme-Selective Detector Systems for High-Pressure Liquid Chromatography," Journal of Chromatography, Vol. 134, pp. 83-90 (1977) proposed the use of a post column reaction zone in the analysis of chromatography effluents containing lactate dehydrogenase isoenzymes. In the Schroeder et al system a chromatographic column effluent was contacted with a solution containing lactate, nicotinamide adenine dinucleotide (NAD) and a buffer. This is the substrate for the reaction catalyzed by lactate dehydrogenase as follows: ##EQU1## In the Schroeder method the NADH (reduced form of NAD) is activated at 340 nm and fluoresces at 457 nm. Two embodiments were proposed. In one embodiment the substrate was added to the column effluent containing lactate dehydrogenase isoenzymes eluted with an NaCl gradient. The effluent stream was split into two segments and passed through respective delay coils (reaction zones), one maintained at 37.degree. C. and one at 18.degree. C. to permit the reactions to occur. The streams leaving the delay coils were each detected by a spectrofluorometer in an effort to provide blank correction. The system, however, was said to be unsatisfactory. Identical flow rates, critical to the blanking, could not be maintained in the split streams because of viscosity differences between the streams due to the different temperatures and to the NaCl gradient. In the second embodiment a substrate containing soluble enzymes was added to the column effluent and passed through two detection zones in series having a delay line (a reaction zone) in between to allow the production of detectable levels of NADH. The delay time between the detectors caused problems which included band spreading. The system was said to be capable of blanking only relatively pure samples not requiring large blank corrections. More complex samples such as human tissue specimens, serum or urine were said to require a computer program to perform matrix manipulation on the first detector output to provide blanking for the output of the second detector. The Schroeder system was also described by Toren, E. C., et al in Abstract 284 of Clinical Chemistry, Vol. 23, No. 6, p. 1172.
Another separation method for isoenzymes has been proposed by Chang, S. H. et al in "High Performance Liquid Chromatography of Proteins," Journal of Chromatography, Vol. 125, pp. 103-114 (1976). The chromatographic column effluent was mixed with soluble substrate and passed through a reaction bed containing an inert support material to permit the formation of spectrophotometrically detectable species. Difficulties could be encountered in the bed of inert material because of physical mixing of bands traveling through the packed column. The system contained no self-blanking apparatus. This application of a post-column reactor bed was also referred to in an abstract, Schlabach, T., et al, "Isoenzyme Analysis by H. P. L. C.," at the Tenth Annual Symposium on Advanced Analytical Concepts for the Clinical Laboratory, Paper No. 8. It can be appreciated from the limitations of the prior art that a simple, accurate, self-blanking system for the analysis of isoenzymes in complex solutions has long been needed.